Reference markers for biological samples

ABSTRACT

DNA oligomers comprising sequences that are absent from the genome of one or more organisms of interest are used as reference markers (RMs). The RMs are added to biological samples to “tag” and subsequently identify the samples as authentic and to distinguish tagged samples from samples obtained without said markers, for example, in forensic, medical, legal and other applications.

This application claims priority to and is a continuation application of U.S. patent application Ser. No. 13/274,592 which is a continuation-in-part of U.S. patent application Ser. No. 12/941,860, filed Nov. 8, 2010. Ser. No. 12/941,860 claims priority to U.S. patent application Ser. No. 11/024,293, filed Dec. 23, 2004 and U.S. Provisional Patent Application 60/532,673, filed Dec. 23, 2003. The complete contents of each of these are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to reference markers (RMs) for biological samples, particularly biological samples that contain or are likely to contain nucleic acids such as DNA. The RMs are oligonucleotides comprising DNA oligomers derived using a system that identifies all sequences of specified lengths that do not exist in (i.e. are absent from) the genome of one or more organisms of interest. The RMs are added to the biological sample in order to “tag” and subsequently identify the sample as authentic, e.g. in forensic, medical, legal or other applications.

BACKGROUND TO THE INVENTION

DNA profiles are routinely used in criminal, paternity, and human identification procedures. The US military requires samples from every soldier, and every state in America requires DNA samples from convicted offenders of qualifying crimes. In addition to these targeted groups, many people are asked to give samples as victims or suspects of crimes.

Reference samples are those given by (or obtained from) known individuals who are part of a forensic, medical, legal or other identification investigation. These samples may be obtained from living or deceased individuals, or from items presumed to be derived from those individuals. A typical example is a blood or buccal sample obtained from a suspect in a criminal investigation.

Once reference samples are obtained, the integrity of the subsequent investigation and analysis depends on the integrity of the reference samples. An investigation can be completely compromised by the cross contamination of reference sample and evidentiary material. There is presently no standard marker added to blood, buccal or other biological reference samples which prevents their accidental or malicious deposition at crime scenes, or in or on evidence samples. In addition, it is not uncommon for the same individuals to handle reference and evidentiary samples. Should cross-contamination occur there is no reliable mechanism of demonstrating that it has happened.

Biological samples are now taken as a standard part of numerous forensic, medical, legal and identification procedures. Several states have enacted legislation defining the length of time that state agencies and forensic laboratories can hold reference samples, but others can hold these samples indefinitely. This has led to a concern on the part of those who provide the samples that errors or malicious intent could lead to their samples being mishandled, thus implicating them in criminal activity. While the DNA in a biological sample serves as an individuating identification of the donor, it says nothing of the manner in which it was obtained. The vast majority of DNA samples are taken as reference samples (known identity), and these must remain separate from evidentiary samples (unknown samples). While several patents address the labeling of samples with chemical markers, none of them satisfy the issues inherent in forensic DNA analysis.

U.S. patent application No. 20040072199 discloses a method for marking samples containing DNA by means of oligonucleotides. This invention does not address forensic applications, and the oligomers disclosed are artificial microsatellites and single nucleotide polymorphisms, designed without reference to avoiding sequences that might be encountered in typical forensic samples.

WO 96/17954 discloses a method for chemical identification of an object, wherein according to the invention at least two chemical markers are used. One marker shows that the container itself has been marked, while the other marker is in principle the real identification. However, such markings are not based on DNA sequences that would be readily detectable using the methodologies common in forensic, paternity and human identification laboratories.

U.S. Pat. No. 5,776,737 discloses a method for the identification of samples, wherein oligonucleotides are added to the sample obtained, which will be sequenced together with the sample after a subsequent amplification step. The oligonucleotides consist of a primer binding site and an identification region consisting of an alternating sequence of nucleotides (MN)x and (MNN)x, respectively, wherein N is the nucleotide of the primer binding site. The sample can be identified by sequencing the identification region. However, this method requires sequencing, and does not address the question of oligomer design in terms of avoiding sequences commonly encountered in forensic samples.

International Patent No. 20030177095 describes a system of authentication and/or tracking for identifying, tracking, authenticating and/or otherwise checking the legitimacy of one or more items which include a coded identity tag or mark, the system comprising identification means for reading said coded identity tag or mark and identifying said one or more items, storage means for storing information relating to the location, whether actual or intended, origin and/or ownership of said one or more items, and means for displaying or otherwise providing or verifying said information relating to an item when its identity tag or mark has been read. However, this system does not cover the specific application of identifying biological reference samples in order to distinguish them from evidentiary samples. It also does not embody a tag that will be identified using the standard techniques in use by forensic, medical, legal and identification laboratories namely Polymerase Chain Reaction and mitochondrial DNA sequencing. Rather it applies to a system which uses tags “preferably in the form of a coded fibre or filament” (claim 3); which can be read by a “bar code reader or scanner” (claim 4). While the claim mentions DNA in its summary (2) as a possible “tag” it does not describe any specific applications or methods using DNA as a tag.

Several patents describe forensic primer sets which are used to amplify human short tandem repeat (STR) regions of the genome. For example, U.S. Pat. No. 6,251,592, for example, discloses (Short Tandem Repeat) STR markers for DNA fingerprinting. This patent is a refinement on the standard technology of DNA fingerprinting for human identification using STR markers.

The prior art has thus far failed to provide a system whereby nucleic acid samples such as forensic reference samples can be marked with amplifiable DNA tags to distinguish the samples from other unmarked samples, thereby establishing their authenticity in a manner compatible with common identification methods such as forensic profiling.

SUMMARY OF THE INVENTION

The invention provides reference markers (RMs), the nucleic acid sequences of which include one or more of the smallest sequences which are absent from a genome or genomes of interest. The absent sequences are identified by analyzing the nucleotide sequence(s) of the one or more genomes of interest using an algorithm that detects sequences that do not occur within the genomic sequence(s). The absent sequences are generally between 11 and 20 nucleotides in length (e.g. 15 nucleotides), but may be longer, and the oligomeric RMs comprise at least one, and usually multiple copies, of one or more absent sequences. The oligomeric RMs are added to biological samples, especially biological samples which are known to contain, or are suspected of containing, genomic DNA or fragments thereof, in order to “tag” or label the samples. As a result, an analysis of the DNA present in a “tagged” sample (e.g. by PCR, sequencing or other means) will reveal the presence of both the RMs and any DNA that was present in the sample. Positive detection of the RM sequences in a sample confirms the history of the sample, and establishes that the sample is authentic. Conversely, if the RMs are not detected in a sample that is purportedly authentic, then the sample may be considered suspect or spurious, either as a result of error, malicious intent or for some other reason. Furthermore, if the RMs are detected e.g. in a forensic sample, which should not contain the RM, then the sample may be considered suspect or spurious, either as a result of error, malicious intent or for some other reason. The RMs are used in conjunction with many applications that involve DNA testing or profiling, for example, in forensic, paternity, archeological and other investigations. In one embodiment, the RMs are used to label and hence distinguish reference from evidentiary samples in forensic investigations. The detection of the RM in evidentiary samples, reagents, or swabs from instruments or work surfaces would indicate contamination of those items. Thus, the RM also serves as a quality control marker for forensic and other DNA laboratories. The RMs also serve as positive amplification control showing that reagents are working properly, and indicating the level of any PCR or profiling inhibitors. Further, if two different sizes of RMs are included in a sample, e.g. in a sample that is stored, detection of differential amounts of the two (e.g. a greater level of loss of a longer RM compared to a level of loss of a shorter RM) may be indicative of the extent of degradation of the sample. In addition, the oligomers or RMs of the invention can serve as virtual or computerized sensing tools for sequences, e.g. for the fidelity of copied data sets, to test for corruption of nucleic acid codes in databases, etc. since detection of the oligomers or RMs of the invention in a data set should not occur. If the oligomer or RM sequences are detected, this is in indication that the data set has been incorrectly copied or corrupted, or is otherwise vitiated. The detection of RM sequences can also detect the introduction of suspected artificial, mutated, or extraterrestrial biological sequences.

The present invention also provides a composition which includes a biological sample that contains at least one oligonucleotide reference marker (RM) as described herein. The reference oligonucleotide sequence does not overlap with a nucleotide sequence found in the genome of a living animal or organism, for example the human genome. In one embodiment the reference oligonucleotide can contain at least one oligomer comprising at least about 11 or 12 consecutive or contiguous nucleotides, and usually at least one oligomer that includes at least about 15 nucleotides, which are not present in the genome of a living animal or organism. In other embodiments, the reference oligonucleotide may contain multiple copies of the unique oligomers described herein, e.g. at least 8 copies of 15 nt oligomers, i.e. at total of 120 or more nucleotides per RM. This invention also provides methods and kits to produce such reference markers for use in forensic, medical, legal, and other applications. The present invention may be utilized in conjunction with many prior art DNA analysis techniques, such as those described in the background section, to insure the identity and authenticity of the samples that are analyzed.

It is therefore an object of this invention to provide reference markers for use in biological samples that do not overlap with the information contained in the biological sample. It is another object of the present invention to provide methods to identify reference markers for use in biological samples that do not overlap with the information contained in the biological sample. It is further object of the present invention to provide kits for use in forensic, medical or other applications that include reference markers that do not overlap with the information contained in the biological sample.

In one embodiment, a method to produce standard reference markers (RMs) to mark and identify biological reference samples. In a second embodiment, a method is provided to add or incorporate the RMs in materials used to collect, transfer and store biological reference samples. In a third embodiment, a method to identify the RMs using techniques that are employed by laboratories involved in identification, processing and analysis of forensic, medical and legal biological reference samples.

In one aspect of the invention, nucleotide sequences are provided that are not found in living organisms, such as in the human genome, especially the smallest of those sequences. A method is provided to generate such sequences by searching the genomes of known organisms. The method includes an iterative search of selected data sets looking for progressively larger sequences not found in the data. Thus, the program looks first for the appearance of each two base combination, then each 3 base combination, etc. The number of possible sequences is represented by the formula 4^(n), where in is the length of the sequence. For example, for an eleven base sequence, the possible number of oligomers is 4¹¹, or 4,194,304. For a 15 base sequence, there are 1,073,741,824 possible combinations. When the program determines that a sequence is not present in the selected data set, it records it as a nullomer.

In one embodiment, a method is provided to distinguish reference samples from those obtained as unknown, questioned or evidentiary samples. This can be achieved because the RM added to the biological reference sample can be detected by PCR, or DNA sequencing.

In one particular embodiment, a Reference Marker (RM) nucleic acid molecule of known sequence and size can be added to reference samples as part of the collection process. The molecules can be included in the containers used to collect, transport and store these samples, such as containers for: buccal swabs, blood, other tissue, and hair samples. The RM can provide a method of distinguishing reference samples from evidentiary, questioned, and unknown samples. RMs can also provide an indicator for tampering with, misidentification of, and misinterpretation of reference samples.

The sequence of the RMs can be formulated so as not to interfere with the commonly used kits for STR analysis. Furthermore, when amplified with primers, the RMs can produce amplicons outside of the range of known human alleles produced by the STR primers in common use by legal, criminal, military, and other human identification laboratories. The RMs also can be formulated so as not to interfere with mitochondrial sequencing. RM primers can be designed in a manner known to those skilled in the art designing STR primers, wherein the primers do not amplify unintended human sequences, or produce amplicons of the reference marker size when combined with materials commonly found in crime scenes.

The RMs can be human “nullomers”. Human nullomers are small sequences which are not present in the human genome. These have been determined by an iterative search algorithm which queries sequences (downloaded to a server) for the complete set of 11, and 12 base sequence possibilities. Based on this analysis 11 and 12 nucleotide base sequences not found in the published human genome sequences can be identified. These sequences that are not found in the genome, we have given the name nullomers. These nullomers can also be searched against the entire set of known sequences in the biosphere, and those sequences that are not found in any species we have given the name “primes.” From the set of nullomers and primes, RMs and their associated primers can be designed.

The RMs can be made of DNA molecules that are either single or double stranded, synthesized oligomers or engineered fragments isolated from vectors. Other nucleotides, nucleotide analogs and organic molecules can be incorporated into the RMs so as compliment STR analysis and sequencing systems.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. Nullomer Tag does not interfere with Quantifiler DUO™ DNA quantification. A, this DNA qPCR quantification standard curve uses control DNA supplied with the Quantifiler DUO™ kit from Applied Biosystems. The DNA standard was diluted according to manufacturer's instructions, and the real-time PCR performed according to the manufacture's protocol. Number of cycles to reach the quantification threshold (Cq) is shown, for DNA with (∘) and without (□) 1.9×10³ copies of nullomer barcode. B, HV2 region of mitochondrial DNA from male (left) and female (right) amplified in the presence and absence of the nullomer barcode. Mitochondrial PCR product was visualized on a 3% agarose gel. HV2 product amplified properly with nullomer barcode (with and without nullomer primers added to the HV2 PCR reaction).

FIG. 2. STR profile of female genomic DNA amplified with Identifiler™ kit in absence of barcode. Size of each amplified product is given in bp, the locus is indicated by labels above the peaks. Labels below peaks indicate the number of repeats (allele) and the relative fluorescence units (RFU) value. The y-axis is in RFUs, and is scaled according to maximum peak height.

FIG. 3. Male DNA amplified with Yplex™ kit in presence of nullomer barcode. No nullomer barcode present in this reaction. The y-axis scale is in relative fluorescence units (RFU). The label for each peak indicates the number of repeats (allele) and the RFU value. Locus DYS385 shows repetition. In Yplex kit nullomer barcode appears as an off ladder peak in the first locus (DYS391).

FIG. 4. DNA extracted from nullomer barcode-impregnated FTA paper, amplified with the Identifiler™ kit. DNA was extracted from nullomer barcode-impregnated FTA paper. Five punches of FTA paper were used for extraction. The y-axis indicates the corresponding RFU value for each peak.

FIG. 5. Human mitochondrial DNA amplified with nullomer barcode. Mitochondrial PCR product was visualized on 3% agarose gel. Presence or absence of target (HV-1) and barcode amplification products are indicated for the different primer combinations used in each reaction (bands at about 500 and 100 bp, respectively).

FIG. 6. Sequence of HV-1 region from female volunteer, from DNA sample tagged with nullomer barcode. 1,900 copies Nullomer barcode added to initial PCR reaction, and another 1,900 copies added to sequencing reactions (Big Dye 3.1 kit, ABI). No nullomer primers were added. The sequences obtained are identical; with nearly identical QV scores for each read (blue bars). Electropherogram detail is shown around the position 16223, where a T is common in the individual's mitochondrial haplogroup (in the Cambridge reference sequence, 16223 is a C).

FIGS. 7A and B. Contamination of evidence with amplified DNA is detected with nullomer barcode, even when diluted 1,000,000 fold. A (and insert), DNA from amplified STR profile (amplified with nullomer barcode) was diluted 100,000 times in water, and then 1 ul of the dilution was applied to a newly purchased knife. This knife was swabbed, and amplified according to the STR kit manufacture's instructions (with the addition of nullomer primers). STR profile from contaminated knife shows both the contaminating profile, and the nullomer barcode peak. B, Electropherogram: original PCR product was diluted 1:1,000,000 in water, and 1 ul of the diluted product was added to a newly purchased knife. The knife was swabbed and processed as above. The nullomer barcode amplicon is prominent, although no alleles from the amplified human profile are detected.

DETAILED DESCRIPTION OF THE INVENTION

The current invention solves a long felt need to ensure the validity and authenticity of samples submitted in forensic, paternity, and other inquiries which utilize DNA analysis in order to establish the identity of a DNA donor. The current invention offers several distinct advantages over standard prior art methods, including but not limited to: the design of reliable, non-dilutable makers suitable for forensic applications, compatibility with standard DNA identification procedures, and a built-in system for laboratory validation concerning the separation of, for example, reference and evidentiary samples. In addition, the methodology is extremely flexible in that a plethora of unique and varied sequences can be designed and tailored to the needs of a particular application.

I: Definitions

The term “RMs” refers to reference markers which are isolated and purified artificial (synthetic) oligonucleotides which comprise at least one copy, and usually a plurality of copies, of a sequence that does not occur (is absent from) one or more genomes of interest. In one embodiment, RMs are added to biological samples (e.g. reference samples) collected from known individuals or sources during the course of forensic, paternity, and other human identification procedures in order to tag or label the samples.

The term “nullomers” refers to oligonucleotide sequences that are not present in the published genome sequences representing a single species.

The term “primes” refers to oligonucleotide sequences that are not present in any reported, published sequence for any species, i.e. these sequences are the smallest (shortest, fewest contiguous nucleotides) that are not present in any sequence of any publicly available biological databases of natural species.

The term “PCR” refers to polymerase chain reaction used to amplify minute amounts of DNA. PCR is a common molecular biology technique in which cycles of denaturation, primer annealing, and primer extension with DNA polymerase, are used to multiply the number of copies of a specific sequence.

The term “amplicons” refers to the amplified products of PCR.

The term “short tandem repeat” (STR) refers to sequences between 2 and 7 nucleotides in length which are tandemly reiterated within the human organism. The STR repeats are usually reiterated between 3 and 50 times.

The term “STR profiling” refers to a length based PCR technique, which is used to identify individuals.

The term “single nucleotide polymorphism” SNP, refers to alternative nucleotide base sequences which differ by a single base. SNPs form the basis of many forms of analysis common in the art.

As used herein, the term “animal” is meant to include any non-human animal, particularly any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovine), deer, mules, horses, monkeys, dogs, cats, rats, mice, birds, chickens, C. elegans, D. melanogaster, reptiles, fish, and insects, including species which are or are thought to be extinct.

II: Determination of Nullomers and Primes

Sequences publicly available on the interne at sites such as the NCBI website can be downloaded and searched using nucleotide sequences of a given length or lengths, for example, the complete set of 11, 12 or 15 base oligomer combinations. For any species, the full set of oligomers that are not found in that species can be termed nullomers. In one embodiment of the invention, nullomers form the basis of the RM sequences to be used to mark reference samples.

In one embodiment, the nullomers described herein can be used as reference markers. In one embodiment, the nullomers can be at least 11, 12 or 15 nucleotides in length. From these 11 or more nucleotide nullomers, oligonucleoitde reference markers of any size can be generated by combining, in a single RM oligonucleotide, at least one copy of one nullomer, or multiple copies of one nullomer, or one copy each of several nullomers, or one or multiple copies of each of several nullomers, or combinations of these. In one embodiment, the reference markers can be at least about 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, lkbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp or more nucleotides in length. In another embodiment, the reference markers can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% homologous to the nullomers.

For example, for human identification RMs, the set of RMs can be derived from the set of 11, 12 or 15 base nullomers determined for the human species. The set of 11 base nullomers derived from two published sequences of the human genome are shown in Table 1. The sequences in bold represent 11 base sequences that are not found in any publicly listed sequence in the NCBI database as determined by BLAST searching on Oct. 12, 2004. These sequences that have not been reported in any species are called “primes”. In one embodiment, the primes are of great value to molecular biology in that they can form the basis of an artificial DNA code, representing sequences that are not found in nature. These sequences are useful as tags which, when detected, indicate the presence of unexpected and possibly synthetic (or at least previously unknown) DNA. This capability could be useful, for example, in investigations of: fossilized remains: samples from locations that are rarely or never-before accessed (e.g. planets other than earth, environments considered to be inhospitable to life such as hot springs, the deep ocean, extreme cold, etc. In addition, the properties and novel consequences of these sequences at the DNA, RNA and protein levels in engineered systems can be exploited as novel features of engineered organisms.

Table 2 below lists 282 15 base human nullomers that are also not found in the known genomes of any other species, i.e. they are primes. These primes were identified as described herein. Briefly, the entire set of publicly available NCBI DNA databases were downloaded, and an iterative search was made of all possible combinations of nucleotides from length 2-17. Absent sequences were first noted at length 15. Counts of all length 15-17 sequences were recorded. The 0 count primes were used to construct the tags described and depicted in the accompanying illustrations.

In a further embodiment of the invention, the list of nullomers and primes can be refined by updated searches of sequence databanks as they are developed. In another embodiment, reference markers can contain at least one nullomer or prime selected from the sequences listed in Tables 1, 2 and 3 below, or their DNA compliments, and/or RNA equivalents. In a further embodiment, the nullomers can be at least 13 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, lkbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp nucleotides in length, which are identified as described herein.

TABLE 1 “Nullomers” not found in the human genome as of 2004. 11 base Nullomers (Human) Sequence SEQ ID NO:  1 cgcgacgttaa 283  2 cgtcgctcgaa 284  3 tacgcgcgaca 285  4 cgcgcataata 286  5 tcgcgcgaata 287  6 cgcgacgcata 288  7 tcgacgcgata 289  8 tcggtacgcta 290  9 gcgcgacgtta 291 10 cgctcgacgta 292 11 cgacggacgta 293 12 tcgcgaccgta 294 13 gtccgagcgta 295 14 cgaatcgcgta 296 15 tgtcgcgcgta 297 16 cggtcgtacga 298 17 cgaatcgacga 299 18 atcgtcgacga 300 19 tagcgtaccga 301 20 gcgcgtaccga 302 21 cgcgtaatcga 303 22 ccgacgatcga 304 23 ctacgcgtcga 305 24 tatcgcgtcga 306 25 cgtatacgcga 307 26 cgattacgcga 308 27 tacggtcgcga 309 28 tattcgcgcga 310 29 cgatcgtgcga 311 30 cgattcggcga 312 31 cgtcgttcgac 313 32 tacgctcggac 314 33 ccgtcgaacgc 315 34 tcggtacgcgc 316 35 taacgtcgcgc 317 36 acgcgcgatat 318 37 ccgcgcgatat 319 38 tcgtcgacgat 320 39 cgacgtaccgt 321 40 ccgacgatcgt 322 41 cgaacggtcgt 323 42 atatcgcgcgt 324 43 cgacgaacggt 325 44 cgcgtatcggt 326 45 tcgacgcgtag 327 46 cgacgaacgag 328 47 cgcgtaatacg 329 48 cgcgctatacg 330 49 tcgcgtatacg 331 50 cgaccgatacg 332 51 gtcgaacgacg 333 52 ttcgagcgacg 334 53 tcgtacgaccg 335 54 tcgcgtaatcg 336 55 tcgccgaatcg 337 56 tcgcacgatcg 338 57 tcgtcgattcg 339 58 tacgcgattcg 340 59 acgaccgttcg 341 60 ccgatacgtcg 342 61 ccgttacgtcg 343 62 acggtacgtcg 344 63 tacgtccgtcg 345 64 accgttcgtcg 346 65 ctcgttcgtcg 347 66 cgtatcggtcg 348 67 tacgtcgagcg 349 68 cgcgtaacgcg 350 69 ccgaatacgcg 351 70 accgatacgcg 352 71 cgtattacgcg 353 72 tcgattacgcg 354 73 cgcgttacgcg 355 74 ttaacgtcgcg 356 75 tatgcgtcgcg 357 76 cgtatagcgcg 358 77 catatcgcgcg 359 78 tattatgcgcg 360 79 cgcgcgatatg 361 80 cgacgtaacgg 362 81 gcgttcgacgg 363 82 cgacgtatcgg 364 83 cgcgtattcgg 365 84 acgatcgtcgg 366 85 tcgatcgtcgg 367 86 atatcgcgcgg 368

TABLE 2 15 base primes as of 2007    1 AACTTCGCTAGCGGG   2 ACCCTAAGGCGCGTA   3 ACCGGGCTAGTCGTA   4 ACCTAGTTCGCGCTA   5 ACGATAGTCTAACGC   6 ACGCGACCGCTAAGT   7 ACGCGCGACTAGTAA   8 ACGGACTAGCGCGCT   9 ACGGTTAGGCCCGTA  10 ACGTAGGGTTACGCG  11 ACGTTAGTACGCCGA  12 ACTAACGTCTCGCGC  13 ACTACGCGTAGGGTC  14 ACTAGCGGTCCGACG  15 ACTAGTACGCTCCCG  16 ACTAGTCGCGGCTAC  17 ACTAGTCGGTACCCG  18 ACTTACGCCCTATCG  19 ACTTACGCGGTCCTA  20 ACTTAGCGGTCGCGT  21 AGCGCGCTAGTCCGT  22 AGCTAGGCGCGTTAC  23 AGGCGCGAACTAGTA  24 AGTCTAACCGGCGTA  25 AGTTAGGCCCGACGC  26 ATACTAGACCGCTCG  27 ATACTAGCGTCGGAC  28 ATAGCCGCGGTCCTA  29 ATAGCGCGTTAGGAC  30 ATCCGACCTAGCGTA  31 ATTAGGCCCGCGATC  32 CATCGGACTAGTACC  33 CCAACCTACGCGTAG  34 CCACGGGCTAGCGTA  35 CCATACGCCTAGTCG  36 CCCCGTACTAGCGGA  37 CCCCGTAGCGAACTC  38 CCCGACTTAAGAGCG  39 CCCGCATACGACTAG  40 CCCGCGTTGTACGTA  41 CCCGCTAGCGAAGTT  42 CCCGGAACTAGCGTA  43 CCCGTATCGCGCTAG  44 CCCGTTACGCGACTA  45 CCCTAACGCGTACTA  46 CCCTACGTCGTAGCG  47 CCCTAGCGACCCGTA  48 CCCTATACGCCGTAG  49 CCGCGTAGGGACTAG  50 CCGGGCGTAGCTAAC  51 CCGGTGTACTAACGC  52 CCGTACTAAGGGCGC  53 CCGTACTACGGCTTA  54 CCTAGGTTCCGCGTA  55 CCTAGTACGACCCGC  56 CCTAGTACGTTACGA  57 CCTAGTATACGCCCG  58 CCTAGTCGCGTAGAC  59 CCTTAGACGCGGTCG  60 CCTTAGTGCGACCCG  61 CGAATCTAGGCGGAC  62 CGACCGCGTCTAAGG  63 CGACTAAGCATACCG  64 CGACTAGGCGTATGG  65 CGAGCGGTCTAGTAT  66 CGATAGGGCGTAAGT  67 CGATAGTCTAACGCG  68 CGATCGGTAACCTAG  69 CGCCTAGTTCCGTAC  70 CGCGAACTTAGTTAG  71 CGCGCGTATTAGACC  72 CGCGGAACGTCCCTA  73 CGCGGAAGTCTAGTA  74 CGCGGACGCTAGTTA  75 CGCGGTCCGTACTAG  76 CGCGTAACCCTACGT  77 CGCGTACGATAGTCC  78 CGCGTAGATTAGTCC  79 CGCGTAGCGGACTTA  80 CGCGTAGGCTAGTTC  81 CGCGTATCGGGCTAG  82 CGCGTTAGACTATCG  83 CGCTACGACGTAGGG  84 CGCTACGTAGTAACG  85 CGCTAGTACGCGAAC  86 CGCTCTTAAGTCGGG  87 CGGACCTACGCGTAA  88 CGGGACTATCCTACG  89 CGGGAGCGTACTAGT  90 CGGGCGTATACTAGG  91 CGGGGGATCGTACTA  92 CGGGGTACGATCTAG  93 CGGGTACCGACTAGT  94 CGGGTCCCGCTAATA  95 CGGGTCGCACTAAGG  96 CGGTACGTACTAGAC  97 CGGTAGGACGCCCTA  98 CGGTATGCTTAGTCG  99 CGGTCGTACTAACCG 100 CGGTCTACGCGTAAC 101

102 CGGTGCGTAGCCCTA 103 CGGTTAGTACGACCG 104 CGTAAGACCGGACCC 105 CGTACGCGGACTAGC 106 CGTACGGCTAACCTA 107

108 CGTAGGACCGTTAAG 109 CGTAGGACGGCCTAA 110 CGTAGGATAGTCCCG 111 CGTAGGGCGTACTTA 112 CGTAGTCCCCGCTAG 113

114 CGTCGCTAATCTAAG 115 CGTCGGACCGCTAGT 116 CGTCGTACTAGGGTC 117 CGTCTAACTAACCGC 118 CGTCTACTAGTCGGA 119 CGTTACTACGTAGCG 120 CGTTAGTACGCGGTC 121 CGTTTAGCGGTCTAC 122 CTAACTAAGTTCGCG 123 CTACGCGTAGGTTGG 124 CTACGGCGTATAGGG 125 CTACGGGGCGTAGTA 126 CTACGGGTAGACCGA 127 CTAGACGCCCGTATA 128 CTAGATCGTACCCCG 129 CTAGCCCGATACGCG 130 CTAGCGCGATACGGG 131 CTAGCGGGGACTACG 132 CTAGCGTACGCTACG 133 CTAGGCGCGATATCC 134 CTAGGTTACCGATCG 135 CTAGTAACTCGCGGC 136 CTAGTACGGACCGCG 137 CTAGTCCCTACGCGG 138 CTAGTCGGACCGTAC 139 CTAGTCGGTACGGGC 140 CTAGTCGTATGCGGG 141 CTAGTCGTCCGGGTA 142 CTCTATAGTCGGACG 143 CTTAACGGTCCTACG 144

145 CTTAGGGCGTTACGC 146 CTTAGTCGCGTCCTA 147 GAACTAGCCTACGCG 148 GACCCTACGCGTAGT 149 GACCCTAGTACGACG 150 GACCGCGTACTAACG 151 GAGTTCGCTACGGGG 152 GATCGCGGGCCTAAT 153 GCCCGACTTAGCGTA 154 GCCCGTACCGACTAG 155 GCCGCGAGTTACTAG 156 GCGCCCTTAGTACGG 157 GCGCGAGACGTTAGT 158 GCGCGATAGGTCTAA 159 GCGCGTCCTATCAAC 160 GCGCTTAGACGGGTA 161 GCGGGTCGTACTAGG 162 GCGGTTAGTTAGACG 163 GCGTAACGCCCTAAG 164 GCGTCCGTAGTCTAC 165 GCGTCGGGCCTAACT 166 GCGTTAGACTATCGT 167 GCGTTAGTACACCGG 168 GCTAGTCCGCGTACG 169 GGACTAATCTACGCG 170 GGACTATCGTACGCG 171 GGATATCGCGCCTAG 172 GGCCGCGTATAGATA 173 GGGCCTAAGTCGCGA 174 GGGCTAGTACGCGTA 175 GGGTACCTAACGCGA 176 GGGTCCGGTCTTACG 177 GGTACTAGTCCGATG 178 GGTCGCGCTACTAGA 179 GGTCTAATACGCGCG 180 GGTTACGCGGACCTA 181 GTAACGCGCCTAGCT 182 GTAACTAACCGCGGA 183 GTAAGACGGGTCGCA 184 GTACGGAACTAGGCG 185 GTACGGGCGCTAGAC 186 GTACGGTCCGACTAG 187 GTAGACCGCTAAACG 188 GTAGACTACGGACGC 189 GTAGCCGCGACTAGT 190 GTCCGACGCTAGTAT 191 GTCCGCCTAGATTCG 192 GTCCGCGCTACGTTA 193

194 GTCGGTACGCCTAGA 195 GTCTACGCGACTAGG 196 GTCTAGACCGCGTTA 197 GTCTAGCGCCCGTAC 198 GTCTAGTACGTACCG 199 GTTACGCGTAGACCG 200 GTTAGACGGCGCGTA 201 GTTAGCTACGCCCGG 202 GTTCGCGTACTAGCG 203 GTTGATAGGACGCGC 204 TAACGCGGTCTAGAC 205 TAACGCGTAAGACCG 206 TAACGTAGCGCGGAC 207 TAACGTCGCGTTAGA 208 TAACTAGCGTCCGCG 209 TAAGCCGTAGTACGG 210 TAAGCTACGGGCGTA 211 TAAGTACGCCCTACG 212 TAAGTCCGCTACGCG 213 TACCCGGACGACTAG 214 TACCCGTCTAAGCGC 215 TACGACTAGCCCGGT 216 TACGCCCGTAGCTTA 217 TACGCCGGTTAGACT 218 TACGCCTAGGGGCGA 219 TACGCGCCGTCTAAC 220 TACGCGCCTTAGGGT 221 TACGCGGAACCTAGG 222 TACGCGTACTAGCCC 223 TACGCTAAGTCGGGC 224 TACGCTAGCCCGTGG 225 TACGCTAGGTCGGAT 226 TACGCTAGTTCCGGG 227 TACGGGCCTAACCGT 228 TACGGGCGTCTAGTA 229 TACGGGGCGTCCCTA 230 TACGGGTCGCTAGGG 231 TACGTACAACGCGGG 232 TACTACGCCCCGTAG 233 TACTAGACGCCCGTA 234 TACTAGACTTCCGCG 235

236 TACTAGTTCGCGCCT 237 TACTTAGGTCCGCGA 238 TAGACCTAGCGCGGA 239 TAGCGCGAACTAGGT 240 TAGCGGACGGTCCTA 241 TAGGACCGCGGCTAT 242 TAGGACCGCGTAAGT 243 TAGGACCGTCCGCTA 244 TAGGACGCGACTAAG 245 TAGGGACGCCCCGTA 246 TAGGGACGTTCCGCG 247 TAGGGCGTCCTACCG 248 TAGGGCTACGCACCG 249 TAGGTCCGCGTAACC 250 TAGGTCTATGCGCGA 251 TAGGTTAGCCGTACG 252 TAGTACGATCCCCCG 253 TAGTACGCCTCCCGA 254 TAGTACGCGTTAGGG 255 TAGTCCGCCCTACGA 256 TAGTCGCGTAACGGG 257 TATACGGGCGTCTAG 258 TATCTATACGCGGCC 259 TATTAGCGGGACCCG 260 TCCGACTAGTAGACG 261 TCCGCGCTAGGTCTA 262 TCCGCGGTTAGTTAC 263 TCCGCTAGTACGGGG 264 TCGAGTCGCCTAGTA 265 TCGCCCCTAGGCGTA 266 TCGCGACTTAGGCCC 267 TCGCGCATAGACCTA 268 TCGCGGACCTAAGTA 269 TCGCGTTAGGTACCC 270 TCGGCGTACTAACGT 271 TCGGGAGGCGTACTA 272 TCGGTCTACCCGTAG 273 TCGTAACGTACTAGG 274 TCGTAGGGCGGACTA 275 TCTAACGCGACGTTA 276 TCTAGGCGTACCGAC 277 TCTAGTAGCGCGACC 278 TGCGACCCGTCTTAC 279 TTACGCGTAGGTCCG 280 TTACTAGTCGCGCGT 281

282 TTAGGCCGTCCTACG (note: the nucleotide compliments of these sequences are also primes). Shading indicates sequences used in the example 2 below. *the number preceding each sequence is the SEQ ID NO: for that sequence.

In another embodiment, the invention encompasses synthetic oligonucleotide primers and probes that hybridize to the oligonucleotides described herein, such as those presented in Tables 1 or 2, or RMs which comprise the oligomers listed in Tables 1 and 2. Those of skill in the art are familiar with techniques for designing primers and probes with properties suitable for use in DNA sequencing or other nucleic acid analysis techniques, including considerations of annealing properties, Tm values, etc. In a preferred embodiment, the primers hybridize under stringent conditions to these oligonucleotides. Another embodiment provides oligonucleotide probes capable of hybridizing to the oligonucleotides described herein, for example, oligonucleotide RMs comprising one or more copies of one or more of the sequences as listed in Tables 1 and/or 2. The polynucleotide primers or probes include at least about 10-50 bases, e.g. at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more bases. In some embodiments, the primers have about 14 bases, or 20 bases, preferably 30 bases or 50 bases which hybridize to a polynucleotide RM of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a preferred embodiment, are at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

The oligonucleotide RMs and the primers and probes of the present invention can be synthesized by any technique known to one skilled in the art. For example, the phosphoramidite method can be used.

III. Construction of RMs

The reference markers of the present invention can be synthesized by any technique known to one skilled in the art. In one embodiment, the primes can be used as a starting material to synthesize a longer reference marker. For example, combinations of various nullomer and/or prime sequences can be generated that can be amplified without interfering with primers used in human identification, and without the risk of amplifying sequences commonly found in evidentiary samples, such as DNA from domestic plants and animals. The RMs for human identification are generally of a length in the range of from about 30 to about 600 nucleotides in length, or from about 70 to about 150 in length, and are preferably of a length of from about 80 to about 120 nucleotides in length. Generally, an RM will comprise from at least about 2 to about 4 prime or nullomer sequences, or from at least about 5 to about 10 prime or nullomer sequences, or from at least about 11 to about 50 or more prime or nullomer sequences. Within an RM, the nullomer and/or prime sequences may be arranged in tandem immediately adjacent to and abutting each other, or the sequences may be separated by linker or spacer sequences. Further, an RM may contain a single (i.e. one) type of nullomer and/or prime sequence (e.g. one sequence from Table 1 or 2, or one sequence repeated several times) or may contain a mix or combination of many different prime and/or nullomer sequences, each of which may be used once or multiple times. In addition, sequences such as restriction enzyme cleavage sties, methylation sites, protein binding sites, transcription promoter sequences, siRNA coding sequences, customized coding information (for date, location, sample type etc), individualized nucleotide tagging sequences, etc., may also be included. Based on the sequences of nullomers and/or primes, RMs can be synthesized for use in conjunction with kits employed in forensic, paternity, human, biotechnology, animal, plant, bacterial, viral and other identification applications. Such kits are well known in the art, and are commercially available from sources such as Applied Biosystems of Foster City, Calif. and Promega of Madison, Wis.

IV. Detection of the RMs

For authenticating or tagging a sample of interest, RMs are added to the sample (or to a container that contains or will contain the sample). In one embodiment 10 to 100 copies of the marker are added to a sample. In another embodiment 100 to 1000 copies of the marker are added to a sample. In another embodiment 1,000 to 10,000 copies of the marker are added to a sample. In another embodiment 10,000 to 100,000 copies of the marker are added to a sample. The RM that is added may be of a single type (i.e. all RMs that are added have the same sequence) or, alternatively, multiple types of RMs with differing sequences may be added to the sample, providing an even higher level of specificity and/or security. Those of skill in the art will recognize that, if multiple types of RMs are used in the practice of the invention, and if PCR amplification is used for detection of the RMs, then multiple primer sets will likely be used for detection, although this need not always be the case, depending on the precise sequence of the RM.

In one embodiment, the RMs will yield amplicons of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 base pairs. In one embodiment the amplicon can be below the size of any common human allele used in STR profiling, for example less than about 90, 80, 70, 60, 50, 40 base pairs. In another embodiment the amplicon can be above the size of any common human allele used in STR profiling, for example more than about 450, 480, 500, 550, 600, 650 base pairs. In a further embodiment, the RM can be detected by DNA sequencing. In another embodiment, the RM can be detected by SNP analysis. In further embodiments, the RM can be identified using PCR, isothermal nucleic acid amplification (such as that used by biohelix, Beverly, Mass.), pyrosequencing, GC/MS, or other methodology.

PCR is based on the use of two specific synthetic oligonucleotides which are used as primers in the PCR reaction to obtain one or more DNA fragments of specific lengths. The test can detect the presence of as little as one DNA molecule per sample, giving the characteristic DNA fragment. Polymerase chain reaction (PCR): a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence by >10⁶ times.

In general, PCR can be performed according to the following protocol (adapted from U.S. Pat. No. 4,683,195). The specific nucleic acid sequence is produced by using the nucleic acid containing that sequence as a template. If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template, either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One physical method of separating the strands of the nucleic acid involves heating the nucleic acid until it is completely (>99%) denatured. Typical heat denaturation can involve temperature ranging from about 80 degrees to 105° C. for times ranging from about 1 to 10 minutes. Strand separation can also be induced by an enzyme from the class of enzymes known as helicases or the enzyme RecA, which has helicase activity and in the presence of riboATP is known to denature DNA. The reaction conditions suitable for separating the strands of nucleic acids with helicases are described by Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLIII “DNA: Replication and Recombination” (New York: Cold Spring Harbor Laboratory, 1978), B. Kuhn et al., “DNA Helicases”, pp. 63-67, and techniques for using RecA are reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37 (1982). If the original nucleic acid constitutes the sequence to be amplified, the primer extension product(s) produced will be completely complementary to the strands of the original nucleic acid and will hybridize therewith to form a duplex of equal length strands to be separated into single-stranded molecules.

When the complementary strands of the nucleic acid or acids are separated, whether the nucleic acid was originally double or single stranded, the strands are ready to be used as a template for the synthesis of additional nucleic acid strands. This synthesis can be performed using any suitable method. Generally it occurs in a buffered aqueous solution, preferably at a pH of 7-9, most preferably about 8. Preferably, a molar excess (for cloned nucleic acid, usually about 1000:1 primer: template, and for genomic nucleic acid, usually about 10⁶:1 primer: template) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand can not be known if the process herein is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also added to the synthesis mixture in adequate amounts and the resulting solution is heated to about 90 degrees-100 degrees Celsius for from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period the solution is allowed to cool to from 20 degrees-40 degrees Celsius, which is preferable for the primer hybridization. To the cooled mixture is added an agent for polymerization, and the reaction is allowed to occur under conditions known in the art. This synthesis reaction can occur at from room temperature up to a temperature above which the agent for polymerization no longer functions efficiently. Thus, for example, if DNA polymerase is used as the agent for polymerization, the temperature is generally no greater than about 45° C. An amount of dimethylsulfoxide (DMSO) can be present which is effective in detection of the signal or the temperature is 35 degrees-40 degrees Celsius. In one aspect of the invention, 5-10% by volume DMSO is present and the temperature is 35 degrees-40 degrees Celsius. For certain applications, where the sequences to be amplified are over 110 base pair fragments, an effective amount (e.g., 10% by volume) of DMSO is added to the amplification mixture, and the reaction is carried out at 35 degrees-40 degrees Celsius, to obtain detectable results or to enable cloning.

The agent for polymerization can be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, and other enzymes, including heat stable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3′ end of each primer and proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths. There can be agents, however, which initiate synthesis at the 5′ end and proceed in the other direction, using the same process as described above.

The newly synthesized strand and its complementary nucleic acid strand form a double-stranded molecule which is used in the succeeding steps of the process. In the next step, the strands of the double-stranded molecule are separated using any of the procedures described above to provide single-stranded molecules.

New nucleic acid is synthesized on the single-stranded molecules. Additional inducing agent, nucleotides and primers can be added if necessary for the reaction to proceed under the conditions prescribed above. Again, the synthesis will be initiated at one end of the oligonucleotide primers and will proceed along the single strands of the template to produce additional nucleic acid. After this step, half of the extension product will consist of the specific nucleic acid sequence bounded by the two primers.

The steps of strand separation and extension product synthesis can be repeated as often as needed to produce the desired quantity of the specific nucleic acid sequence. As will be described in further detail below, the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion.

When it is desired to produce more than one specific nucleic acid sequence from the first nucleic acid or mixture of nucleic acids, the appropriate number of different oligonucleotide primers is utilized. For example, if two different specific nucleic acid sequences are to be produced, four primers are utilized. Two of the primers are specific for one of the specific nucleic acid sequences and the other two primers are specific for the second specific nucleic acid sequence. In this manner, each of the two different specific sequences can be produced exponentially by the present process. The polymerase chain reaction process for amplifying nucleic acid is described in, for example, U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202, the complete contents of each of which are hereby incorporated by reference, and European patent Nos. EP 201184 EP 200362.

DNA samples are subjected to PCR amplification using primers and thermocycling conditions specific for each locus that contains a sequence of interest, e.g. an RM as described herein. In one example, the primers are selected from the group of sequences shown in Tables 1 and 3. The specific amplification procedures and primer sequences relating to each locus and allelic ladder, as well as a description of locus-specific primers are described in U.S. Pat. Nos. 6,156,512 and 5,192,659, the complete contents of each of which are hereby incorporated by reference.

V. Application of RMs to Substrates and Containers

In one embodiment, the RMs can be added to a solid substrate or container, for example, the collection substrates of kits used for sample collection, such as in forensic or medical applications. Such kits are available in a number of forms and include various substrates for samples. The RM molecules can be added directly to a component of the kit which is suitable for receiving a nucleic acid sequence. This component is generally the same as or similar to a component that will also receive the unknown DNA sample that is being authenticated. One exemplary kit is the FTA® classic card, manufactured by Whatman, plc, Brentford, Middlesex, UK. Kits of this type include FTA® paper to which the RMS may be added, either during manufacture or subsequently. The RMs can be applied as an aqueous solution, powder, gel, laminate, spray, resin, etc. or in a form such as a capsule, or in any other suitable form. RMs may also be coated or spotted onto the walls of a collection container, or impregnated into a swab or other component of a kit.

In another embodiment, the RMs can be added e.g. to a liquid in the collection vessel such as the Vacutainer System of Becton, Diskinson and Company, Franklin Lakes, N.J.

In another embodiment, the RMs can be combined with agents or processes used in sample preparation, storage or processing, e.g. agents that are used to protect DNA samples at room temperature.

VI. Kits

In other embodiments of the present inventions, kits are provided that include the oligomers and/or reference markers of the present invention. In addition, the kits can include applicator sticks, swabs, tubes, membranes, cotton, nylon, FTA® paper, locking mechanisms, vessels, chambers, buffers, fixatives, drying agents, labels, bar codes, needles, microneedles, pins, lances, anticoagulants, EDTA, heparin, preservatives, primers, magnesium, DTT, dyes, antibodies, alcohol, extraction buffer, phenol, chloroform, proteinase K, SDS, etc. or any other suitable components.

The invention thus also provides kits containing a reference oligonucleotide marker, wherein the oligonucleotide sequence of the RM does not overlap with a nucleotide sequences reported in the human genome (or other genome(s) in publically available databases of natural species), which is deposited in or on a container. In one embodiment the kit also contains a self-locking system, wherein the swab used for buccal scraping is broken off from the applicator stick and deposited in a self sealing tube which contains the reference marker.

The RMs described herein may be used for any of a number of applications in which nucleic acid analysis is involved. The nucleic acid analysis is frequently DNA sequencing. The applications include but are not limited to, for example: forensic/crime investigations; paternity investigations; medical applications; archeological investigations; verifications of the identity of living or deceased subjects or of remains of subjects; investigations of genealogical bloodlines (e.g. of “prize” animals, or of the ancestry of a human or humans, either recent genealogies or so-called “deep” ancestry extending into prehistoric times; testing of food or other items for contamination (e.g. for a source of food poisoning, for chemical contaminants, etc.) or for any investigation which involves the collection of nucleic acid samples or samples and their subsequent analysis, especially in cases where it is important to verify or authenticate or track the origin of a sample, e.g. to establish that the sequence of a sample is accurately attributed to or associated with the actual source of the sample. Generally, samples that are analyzed using the methods described herein are known to contain or suspected of containing genomic DNA from an organism or individual of interest, although this may not always be the case. Other applications for this technology also exist, including but not limited to: the use of the oligomers to authenticate samples intended for analysis with respect to contamination, e.g. by microbes, such as water, food samples, etc.; the use of the oligomers to authenticate samples of substances which are intended for analysis with respect to chemical contamination; the use of the oligomers to label and trace or track manufactured goods (e.g. synthetic oligonucleotides, synthetic genes, synthetic genomes or portions thereof, vectors, etc.); samples of naturally occurring or genetically engineered organisms (bacteria, algae, fungi, cultured cells, etc.); or articles or items that have nothing to do with nucleic acids per se (e.g. food items, various liquids, textiles, household goods, etc.), providing the manufacturing and storage or use of the product does not destroy the integrity of the RMs, and so long as the RMs remain detectable in a reliable manner within or associated with the product.

EXAMPLES Example 1 Identification of 11 and 12 Base Nullomers

The human genome has been searched using an iterative algorithm which looks for the smallest sequences not found in the selected genome. Our results are presented in Table 1 for the two publicly available human genome sequences. The oligomer sequences not found in the selected genome are called nullomers. The complete set of 11 and 12 base nullomers for the human genome have been determined using this method. These sequences can be used to construct artificial genomes, or genetic elements such as tags, novel protein epitopes, and novel RNA sequences and structures, not found in the human genome. The human nullomers were then used for BLAST searches with the goal of identifying: those sequences which were not represented in any living organism, those that were rare (represented less than 5 times in all the publicly available sequences), those not found in mammals, those not found in eukaryotes, those not found in viruses, those not found in plants, those not found in bacteria; and those not found in combinations of these organism groups. These sequences can be used to construct artificial genomes, or genetic elements such as tags, novel protein epitopes, and novel RNA sequences and structures, not found in the known sequences of the biosphere.

In one embodiment, the reference markers (RMs) will yield amplicons of about 60 base pairs, below the size of any common human allele found in STR profiling. One example of an amplicon generated by amplification of an RM-based nullomer tag is cgacgtatcgg accgttcgtcg ccgatacgtcg cgacgaacggt tacgctcggac gtccgagcgta cgctcgacgta (SEQ ID NO: 369).

Example 2

Unintended transfer of biological samples is an issue of great concern to all laboratories conducting sensitive analysis. This is particularly true for crime laboratories, where victims and suspects are asked to supply reference samples of their DNA for comparison to evidence profiles. Contamination, or unintended transfer, can happen at any time in forensic DNA analysis, from before sample collection until entry into the database. Instances of unintended transfer, and mislabeling by personnel collecting and processing samples, are well documented. This problem is likely to become more pronounced as forensic DNA techniques become ever more sensitive. Laboratories which use enhanced techniques for low template DNA analysis have detected contamination in reagents, plastic ware and laboratory samples at levels that are below detection with less sensitive methods. Likewise, secondary and tertiary transfer of DNA that was previously below detection limits is now more readily seen with techniques that can produce profiles from just a few cells. Finally, because laboratory protocols amplify molecular targets millions of times over, extreme caution must be taken to isolate amplified DNA from all areas where evidence processing occurs. Even a million-fold dilution of amplified DNA contains enough template in a fraction of a microliter to cause contamination.

In order to safeguard against the accidental or malicious transfer of DNA samples collected from members of the public, we have developed a universal tag that can be modified to code a variety of information, such as laboratory location, testing purpose, or date. These tags are based on the smallest sequences absent from all publicly available DNA databases (nullomers). The nullomer approach has generated interest in algorithms for counting and tracking biological sequences, and several researchers have recently proposed efficient methods for determining the set of shortest absent sequences, and the set of minimal absent sequences. The algorithm used to identity the nullomers and primes described herein is able to process the entire set of biological sequence data found on NCBI's web site in less than 8 hours, calculating the frequencies of all sequences up to length 17 (longer lengths can be calculated as well). At length 17 there are (as of January 2011) approximately 700 million (695,038,288) absent sequences.

This Example describes the first practical application of nullomer sequences. The results show that nullomer sequences can be used as molecular barcodes, successfully integrated into the multiplex PCR reactions of commercially available forensic profiling kits, and used along with PCR for sequencing.

Methods

Nullomer Barcode Identification

Candidate nullomer sequences for barcodes were identified by examining all of the DNA sequence data available from the National Center for Biotechnology Information (NCBI) website using a robust software algorithm we developed for this purpose (Hampikian, G. & Andersen, T. Absent sequences: nullomers and primes. Pac Symp Biocomput, 355-366 (2007); the complete contents of which are hereby incorporated by reference). The algorithm, which is capable of processing the entire data set in about 8 hours, counts the number of possible sequences up to a maximum specified length, and outputs the sequences that have a zero count. Currently, there are approximately 700 million DNA 17-mers that have a zero count, i.e. are not found in any of the DNA data available from NCBI (and 2,358,580 absent 16-mers as well as 34 absent 15-mers), which makes it possible to construct an enormous number of unique DNA based tags using relatively short sequences.

Nullomer Barcode Construction

To construct the barcodes for our experiments, eight 15-mer nullomer sequences were arranged in tandem. Sequences were chosen to minimize the formation of secondary structure, by running sequences in the OligoAnalyzer tool on the Integrated DNA Technologies (IDT) website located at www.idtdna.com. Terminal sequences (primer binding regions) were chosen so that the annealing temperature would match as closely as possible the 59° C. annealing temperature of the ABI kit protocols (58° C. for Powerplex-Y kit). The 120 bp construct was synthesized by IDT (Coralville, Iowa, USA) as two complimentary single stranded molecules. This complimentary pair was annealed to make it double stranded, using a thermal gradient from 95° C. to 75° C. After annealing the nullomer strands, ExoSAP™ enzyme treatment was used to cleave all the remaining single stranded molecules. Primers were designed to yield amplicon sizes of 88, 90, and 114 bp. Several barcode concentrations were tested with different STR kits; results are presented using 1,900 copies per PCR reaction, which gave barcode peaks at intensities comparable to the human STR alleles.

Real-Time PCR Quantification

For DNA quantification a DNA dilution series was made, according to the manufacturer's protocol, with the male genomic DNA standard supplied with the Duo kit. The nullomer barcode (without barcode primers) was added to each of the qPCR reactions. PCR was performed on an Eppendorf Mastercycler realplex instrument and data analyzed with Realplex software and GraphPad Prism 5 software.

STR and Y-STR Amplification

The compatibility of nullomer barcodes was tested with STR kits from ABI (Identifiler™, Yfiler™, Profiler Plus™) and Promega (Yplex™). The Profiler Plus™kit amplifies 10 loci, and the Identifiler amplifies 16 loci (including amelogenin). The Yfiler™ kit amplifies 17 Y-STR loci, and Yplex™ amplifies 11 Y-STR loci. For STR reactions, ABI and Promega's standard protocols were strictly followed, e.g. thermal cycling, reaction buffer, and primer mix concentrations. Approximately 1,900 nullomer barcode amplicons were added to each 1 ng human DNA samples extracted from buccal swabs. Barcode primers were at 2.5 μM final concentration per PCR reaction. The amplicons were analyzed in an ABI 3130 genetic analyzer, with Pop-4™ polymer, using Gene Mapper ID-X software.

Human Mitochondrial DNA Sequencing

Three different concentrations of nullomer barcode molecules were added to sequencing reactions: 962, 1900, and 3800 copies. Amplicons were visualized on a 3% agarose gel to verify both bands present (HV1 and HV2), along with the nullomer barcode bands. For sequencing reactions, nullomer barcodes were added to the reactions but not the barcode primers. Pop-4™ polymer and Big Dye 3.1® terminator chemistry were used for sequencing. PCR products were purified by ExoSap (USB) kit and also with the Bigdye® XTerminator™ kit (ABI) before sequencing. 3-10 ng DNA were used for each cycle sequencing reaction. Both the HV1 and HV2 control regions were analyzed to verify that the nullomer barcode did not interfere with mitochondrial sequencing reactions.

DNA extraction from swab and FTA paper

Approximately 1.5×10⁸ copies of the barcodes were dissolved in 100 μl of double distilled water applied to 572.6 mm² FTA paper (Fitzco Inc, Minnesota, USA), and allowed to dry overnight. Then cells from a male and female donor's buccal swabs were applied to the FTA paper by gently rubbing each swab against the card. Five punches (2 mm diameter) were taken from each FTA card. Assuming an even distribution of nullomer barcode solution, we estimate that each punch had approximately 1.5×10⁷ copies of the nullomer barcode. DNA extraction was performed using Qiagen's DNA Mini kit (according to manufacturer's protocol). The amount of total extracted DNA was quantified on a Nanodrop ND-1000. Quantification was visually checked by agarose gel electrophoresis with Lonza's quant ladder, and visualized with a Gel Doc XR imager (BIO-RAD) using Quantity One 4.6.5 software (BIO-RAD). One nanogram of extracted DNA was used to generate a profile using the Identifier kit supplemented with forward and reverse barcode primers.

Mock “unintended transfer” of amplified DNA to a knife in the laboratory DNA was extracted from a buccal swab (Whatman sterile OmniSwab), transferred to FTA paper containing the nullomer barcode (as above) and amplified with ABI's Identifiler kit. All reactions were processed according to manufacturer's instructions with the addition of the nullomer primers to the PCR reaction. After STR amplification, 1-10 μl of post amplified DNA was applied to several knives to see the abundance and stability of nullomer barcodes. Post PCR reactions were also diluted 100,000, and 1,000,000 times before applying to the knife and swabbing. Each knife was swabbed with a sterile omniswab, and DNA was extracted with Qiagen's DNA mini kit using a 15 minute, 56° C. incubation in the extraction buffer. The extracted DNA was amplified with the Identifiler™ kit.

Sequences for Nullomer RMs (barcodes) and primers used in this study were as follows:

Nullomer Barcode (SEQ ID NO: 370) 5′-TAC TAG GCG ACT CGA CGG TCT TAC GCG TTA CGT CCG ACT ATA GAG CTT AGA TTA GCG ACG TTA GAC CTA TCG CGC CTT AGA TTA GCG ACG CTA GCG TAC GCT ACG GTC CTA ACG CGC TAT-3′; Primer_114F (SEQ ID NO: 371) 5′-TAG GCG ACT CGA CGG TCT TAC GCG TTA CGT; Primer_114R (SEQ ID NO: 372) 5′-GCG CGT TAG GAC CGT AGC GTA CGC TAG CGT; Primer_88F (SEQ ID NO: 373) 5′-TAC TAG GCG ACT CGA CGG TC; Primer_88R (SEQ ID NO: 374) 5′-TCG CTA ATC TAA GGC GCG ATA GGT C; Primer_90F (SEQ ID NO: 375) 5′-TAC TAG GCG ACT CGA CGG TC; Primer_90R (SEQ ID NO: 376) 5′-CGT CGC TAA TCT AAG GCG CGA; Prime 108 R  (108 by product when paired with Primer F_114) (SEQ ID NO: 377) 5′-TAG GAC CGT AGC GTA CGC TAG CGT CGC TAA. Results

Adding the nullomer tag to a human buccal swab did not affect the quantification of extracted DNA (FIG. 1A). The kit used in our experiment (Quantifier® Duo) is commonly employed by forensic laboratories to determine the DNA concentration for both the total human and male fraction (Y-chromosome) of a sample. The data presented here shows that the nullomer tag does not negatively impact either quantification. We designed the barcode DNA amplicons to be a size outside the range of human alleles, so that there is no confusion between the barcode and known STR allele peaks. When amplified with the STR alleles of the forensic kits we tested, a 90 base pair nullomer barcode appears in the electropherogram as an additional peak outside the first bin set. Human DNA and barcode DNA was amplified according to manufacturer protocols (except for the addition of the nullomer barcode and barcode primers) with several standard techniques. Exemplary results obtained with Identifiler™ are presented in FIG. 2. Similar successful results were obtained using Profiler Plus™, Yfiler™, and YPlex™ kits. A number of different nullomer-based primer sets have been created for applications that may benefit from larger or smaller tags. The results for an 88 base pair and a 114 base pair amplicon using the Identifiler™ kit showed that amplification was successful and the barcod RMs were readily distinguishable (not shown) We also demonstrated that the addition of a nullomer-specific primer set is required, and that the nullomer barcode cannot be amplified with STR primers alone (FIG. 3). Therefore, the addition of these barcodes does not affect the typical analysis of forensic DNA.

A common technique for the storage and processing of reference samples is to transfer a buccal swab sample to FTA paper and allow it to dry. DNA samples fixed onto FTA paper can be stored at room temperature, and then be extracted from FTA paper punches when needed. When we extracted and amplified DNA from nullomer-treated FTA paper, we obtained a profile which includes the barcode tag, identifying the DNA as coming from a reference source (FIG. 4), and not from evidence.

In order to test whether our nullomer tag is compatible with mitochondrial DNA (mtDNA) sequencing, we amplified the HV-1 and HV-2 regions of human mtDNA in the presence of nullomers, with and without tag primers. The barcode PCR products were detected as distinct bands in a 3% agarose gel (FIG. 1B and FIG. 5). Mitochondrial DNA sequencing was not adversely affected by the presence of the nullomer barcode, whether the barcode was added during initial PCR using HV1 and HV2 primer sets, or during sequencing reactions using HV1 or HV2 amplicons as templates (FIG. 6).

We setup a mock unintended transfer of Identifiler amplified DNA (with barcode) to an evidentiary weapon (knife). A 105-fold dilution of the PCR product showed clear amplification of nullomer barcode DNA along with a partial profile of the transferred human amplicons (FIG. 7A). After a 106-fold dilution, the nullomer barcode could be detected; even though the human profile was lost (FIG. 7B).

We have shown that an artificial DNA barcode can be used in conjunction with forensic genetic analysis kits, without affecting DNA quantification, STR amplification, profile determination, or mitochondrial sequence, using standard protocols. DNA profiles were obtained by amplification of 0.5-1.0 nanograms of genomic DNA in the presence of 500-8000 copies of nullomer barcodes. The nullomer barcodes can used to pre-treat FTA paper, and we envision nullomer-tagged FTA kits, which would mark samples without affecting forensic DNA profiling.

Extrinsic DNA can enter the laboratory through contaminated reagents, disposables, centrifuges and water baths. Reagent controls and routine monitoring can detect many of these examples, however, contamination of evidence with DNA from reference samples taken from suspects, or the switching of reference samples, is more difficult to detect, and may in fact implicate an innocent person in a crime. The nullomer tags described in this paper were developed to assure the public that their reference samples can be marked so as to prevent false incrimination. Further development of the tag technology can be used to code individual samples to further safeguard the public. The initial tags described here can be added to collection kits so that reference samples are safeguarded from the point of collection, through handling by the collector, laboratory processing and storage, amplification, and even post amplification. Physical and chemical modifications of the tags could be used to stabilize them further; though we demonstrate here that even unmodified synthetic DNA is sufficient. While the barcode is not designed to detect environmental contamination, it will detect contamination or mislabeling involving reference and evidence samples processed in the same lab. The barcodes have been stored at 4° C. for one year in TE buffer without affecting amplification and detection, and used FTA paper with dried barcodes has been used for six months without any noticeable effect on amplification.

Synthetic DNA barcoding has been used to make positive amplification controls for applications in a wide variety of fields, such as clinical microbiological testing and food pathogen testing, and has been discussed for a broad variety of tagging and tracing protocols; but unlike nullomer tagging these methods do not build their artificial sequences from combinations of the smallest sequences absent from public databases. While these sequences may eventually appear in a natural database, it is highly unlikely that concatamers based on them will. The probability that such a sequence will arise and work with primers designed specifically for the barcode primers, and produce the proper sized amplicons, is extremely small. Even if this does occur, that sequence would have to be present in an evidentiary sample to be problematic (and sequencing the DNA from the sample would reveal this coincidence).

The exemplary nullomer barcode described herein is 120 bases long, and since only 1,900 copies are needed per PCR reaction, the additional cost to sampling kits would be minimal. A single 100-μg synthesis (less than $500) is sufficient for more than a million forensic tests. These barcodes could easily be added to FTA paper, liquid buffer, cotton swabs, or other components of human DNA sampling kits. While DNA has the power to free the wrongfully convicted, it can also be the route of forensic error as illustrated by a recent, highly publicized wrongful conviction and incarceration, due to mislabeling of DNA samples. As DNA sampling and archiving becomes routine, the public needs to be assured that their DNA is being properly collected, stored and interpreted, and the present invention provides such a safeguard. 

The invention claimed is:
 1. A method of making and using a kit for authenticating or tracking a biological sample of a human or other species, comprising the steps of: synthesizing one or more reference markers comprising one or more nucleic acid oligomers selected from the group consisting of SEQ ID NOs: 1-368; and providing said one or more reference markers for use as an authenticating or tracking molecule in said biological sample; combining said biological sample with said one or more reference markers; and then determining if a test sample includes said one more reference markers, wherein if said test sample includes said one or more reference markers then said test sample is authenticated to be said biological sample, and if said test sample does not include said one or more reference markers then said test sample is determined to not be said biological sample.
 2. The method of claim 1, wherein said biological sample is of a human.
 3. The method of claim 1, wherein said biological sample is a forensic sample.
 4. The method of claim 1, wherein said one or more nucleic acid oligomers are selected from the group consisting of SEQ ID NOs: 283-368.
 5. The method of claim 1, wherein said one or more nucleic acid oligomers are selected from the group consisting of SEQ ID NOs: 101-282. 